Huijun Yuan

GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment

BackgroundMutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI). The mutation spectra of this gene vary among different ethnic groups.MethodsIn order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced.ResultsA total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X) are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles) varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups.ConclusionIn some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary.

A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China.

There is a worldwide interest in studying SLC26A4 mutations that are responsible for enlarged vestibular aqueduct (EVA) in different ethnic background and populations. The spectrum of SLC26A4 mutations in Chinese population is yet to be fully characterized. In this study, all the 21 exons of SLC26A4 were screened in 107 Chinese patients with hearing loss associated with EVA or both EVA and Mondini dysplasia (MD), taken from six multiplex and 95 simplex families. The two types of control populations consisted of 84 normal‐hearing subjects and 46 sensorineural hearing loss subjects without inner ear malformations. Biallelic mutations were found in 12 patients from multiplex families and 84 patients (88.4%) from the simplex families. In addition, monoallelic variant was detected in nine patients in the remaining 11 simplex families. Overall, up to 97.9% patients were found having at least one possible pathogenic variant in SLC26A4, with most having biallelic variants consistent with recessive inheritance of this disorder. A total of 40 mutations including 25 novel mutations were identified in the Chinese patients but were not detected in all the controls except for one normal subject. For the Chinese mutation spectrum of SLC26A4 gene, IVS7‐2A>G mutation was the most common form accounting for 57.63% (102/177) of all the mutant alleles.

Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China

BackgroundEvery year, 30,000 babies are born with congenital hearing impairment in China. The molecular etiology of hearing impairment in the Chinese population has not been investigated thoroughly. To provide appropriate genetic testing and counseling to families, we performed a comprehensive investigation of the molecular etiology of nonsyndromic deafness in two typical areas from northern and southern China.MethodsA total of 284 unrelated school children with hearing loss who attended special education schools in China were enrolled in this study, 134 from Chifeng City in Inner Mongolia and the remaining 150 from Nangtong City in JiangSu Province. Screening was performed for GJB2, GJB3, GJB6, SLC26A4, 12S rRNA, and tRNAser(UCN)genes in this population. All patients with SLC26A4 mutations or variants were subjected to high-resolution temporal bone CT scan to verify the enlarged vestibular aqueduct.ResultsMutations in the GJB2 gene accounted for 18.31% of the patients with nonsyndromic hearing loss, 1555A>G mutation in mitochondrial DNA accounted for 1.76%, and SLC26A4 mutations accounted for 13.73%. Almost 50% of the patients with nonsyndromic hearing loss in these typical Chinese areas carried GJB2 or SLC26A4 mutations. No significant differences in mutation spectrum or prevalence of GJB2 and SLC26A4 were found between the two areas.ConclusionIn this Chinese population, 54.93% of cases with hearing loss were related to genetic factors. The GJB2 gene accounted for the etiology in about 18.31% of the patients with hearing loss, SLC26A4 accounted for about 13.73%, and mtDNA 1555A>G mutation accounted for 1.76%. Mutations in GJB3, GJB6, and mtDNA tRNAser(UCN)were not common in this Chinese cohort. Conventionally, screening is performed for GJB2, SLC26A4, and mitochondrial 12S rRNA in the Chinese deaf population.

Cosegregation of the G7444A Mutation in the Mitochondrial COI/tRNASer(UCN) Genes with the 12S rRNA A1555G Mutation in a Chinese Family with Aminoglycoside-induced and Nonsyndromic Hearing Loss

We report here on the characterization of a three‐generation Chinese family with aminoglycoside‐induced and nonsyndromic hearing impairment. Ten of 17 matrilineal relatives exhibited bilateral and sensorineural hearing impairment. Of these, nine matrilineal relatives, who had a history of exposure to aminoglycosides, exhibited variable severity and audiometric configuration of hearing loss. The dose and age at the time of drug administration seemed to be correlated with the severity of the hearing loss experienced by affected individuals. Sequence analysis of the complete mitochondrial genome in the pedigree showed the presence of homoplasmic A1555G mutation and 37 variants belonging to haplogroup D4a. Of those variants, the G7444A mutation is of special interest as the mutation at this position results in a read‐through of the stop condon AGA of the COI message, thereby adding three amino acids (Lys–Gln–Lys) to the C‐terminal of the polypeptide. Alternatively, the G7444A mutation is adjacent to the site of 3′ end endonucleolytic processing of L‐strand RNA precursor, spanning tRNASer(UCN) and ND6 mRNA. Thus, the G7444A mutation, similar to the deafness‐associated A7445G mutation, may lead to a defect in the processing of the L‐strand RNA precursor, thus influencing the phenotypic expression of the A1555G mutation. These data also imply that nuclear background plays a role in the aminoglycoside ototoxicity associated with the A1555G mutation in this Chinese pedigree.

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.

Mutation of the ATP-gated P2X2 receptor leads to progressive hearing loss and increased susceptibility to noise

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X2 receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X2 receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X2 function is required for life-long normal hearing and for protection from exposure to noise.

Loss-of-Function Mutations in the PRPS1 Gene Cause a Type of Nonsyndromic X-linked Sensorineural Deafness, DFN2

We report a large Chinese family with X-linked postlingual nonsyndromic hearing impairment in which the critical linkage interval spans a genetic distance of 5.41 cM and a physical distance of 15.1 Mb that overlaps the DFN2 locus. Mutation screening of the PRPS1 gene in this family and in the three previously reported DFN2 families identified four different missense mutations in PRPS1. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients. By in situ hybridization, we demonstrate expression of Prps1 in murine vestibular and cochlea hair cells, with continuous expression in hair cells and postnatal expression in the spiral ganglion. Being the second identified gene associated with X-linked nonsyndromic deafness, PRPS1 will be a good candidate gene for genetic testing for X-linked nonsyndromic hearing loss.

Variants in mitochondrial tRNAGlu, tRNAArg, and tRNAThr may influence the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese families with hearing loss†

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside‐induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing loss including severity, age‐at‐onset, audiometric configuration in these subjects. Penetrances of hearing loss in BJ107, BJ108, and BJ109 pedigrees are 35%, 63%, and 67%, respectively. Mutational analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mitochondrial DNA (mtDNA) variants belonging to haplogroups N, F, and M, respectively. Of these variants, the A14693G mutation in the tRNAGlu, the T15908C mutation in the tRNAThr, and the T10454C mutation in the tRNAArg are of special interest as these mutations occur at positions which are highly evolutionarily conserved nucleotides of corresponding tRNAs. These homoplasmic mtDNA mutations were absent among 156 unrelated Chinese controls. The A14693G and T10454C mutations occur at the highly conserved bases of the TψC‐loop of tRNAGlu and tRNAArg, respectively. Furthermore, the T15908C mutation in the tRNAThr disrupts a highly conserved A‐U base‐pairing at the D‐stem of this tRNA. The alteration of structure of these tRNAs by these mtDNA mutations may lead to a failure in tRNA metabolism, thereby causing impairment of mitochondrial translation. Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA mutations. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness‐associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss

Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and nonsyndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss, using denaturing HPLC (DHPLC) and direct DNA sequencing. Fifty-two of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas SLC26A4 mutations were found in only 2 of 116 deaf Chinese patients without EVA. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573) and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl(-)/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wild-type levels. Two variants, p.F354S and p.E737D, displayed selective reduction in relative rate of Cl(-)/HCO(3)(-) exchange compared with similarly measured rates of Cl(-)/Cl(-) and Cl(-)/I(-) exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.

The prevalence of the 235delC GJB2 mutation in a Chinese deaf population

Purpose: Mutations in the GJB2 gene are the most frequently found mutations in patients with nonsyndromic hearing impairment in populations studied to date. However, the prevalence of mutations varies among different ethnic groups. In most areas of China, genetic testing for nonsyndromic hearing impairment is currently not available because of the lack of information regarding the molecular cause of nonsyndromic hearing impairment. The purpose of this study is to determine the prevalence of a common GJB2 mutation, 235delC, in Chinese deaf children.Methods: We collected DNA specimens from 3004 patients with nonsyndromic hearing impairment from 26 regions of China; 368 Han Chinese and 98 Uigur controls, and screened for the 235delC mutation. The coding exon of the GJB2 gene was polymerase chain reaction amplified, followed by restriction enzyme digestion with ApaI and analysis by agarose gel.Results: Overall, 488 patients (16.3%) were determined to carry at least one 235delC mutant allele, with 233 (7.8%) homozygotes and 255 (8.5%) heterozygotes. Therefore, within the subpopulations examined, the frequency varies from 0% to 14.7% for 235delC homozygotes and from 1.7% to 16.1% for heterozygotes. On the basis of this survey of the patient cohort as stated, Chinese patients with nonsyndromic hearing impairment appear to have a relatively higher 235delC frequency than that of other Asian populations.Conclusion: These results demonstrate that an easy and fast genetic testing method for this well-known GJB2 gene mutation can be made available for at least 2 million Chinese patients and family members with nonsyndromic hearing impairment. By screening for the common GJB2 235delC mutation, the molecular cause in as high as 15% of patients with nonsyndromic hearing impairment in certain regions of China can be identified. In addition, patients who are negative for the 235delC mutation would be candidates for further mutational analysis of GJB2 or other deafness-related genes.